increasingly utilized as a rapid technique to identify microorganisms by their molecular fingerprint and/or by biomarker detection and it represents a first-line method for the accurate routinely identification of bacteria and fungi; its application in parasitology is on the contrary very limited. In this study MALDI-TOF MS was used to identify Trichomonas vaginalis, Dientamoeba fragilis and to differentiate Entamoeba histolytica and E. dispar. Introduction Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is Materials and Methods In this study, an aliquot of one cultured reference strain for each parasite were submitted to formic acid/acetonitril protein extraction and to MALDI-TOF MS analysis. The spectrum obtained for T. vaginalis was suplemented in the Bruker Daltonics database (Bruker Daltonics, Germany) and a new identification method was created by modifying the range setting for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. The spectra obtained for E. histolytica, E. dispar and D. fragilis were analysed and subsequently imported into the ClinProTools software version 2.2. (Bruker Daltonics) to perform a statistical analysis in order to check the presence of specific peaks for each parasite. To verify the reliability of the system 21 T. vaginalis, 6 E. histolytica, 8 E. dispar and 13 D. fragilis clinical isolates, respectively, were used. Results After implementation and modification of the paramenters' setting, the protein spectra of T. vaginalis clinical isolates were correctly identified. Five discriminating peaks between E. histolytica (2 peaks) and E. dispar (3 peaks) and 6 discriminating peaks for D. fragilis were found, respectively. When the spectra belonging to the clinical isolates were analysed, all the identifications matched those obtained by a specific Real-time PCR, except for one E. histolytica strain. Dicussion and Conclusions Although the massive number of entries in the available commercial database, the absence of reference spectra of parasites does not allow their identification. Our study demonstrated that MALDI-TOF MS can be applied to the identification of parasites by using two different approaches: i) the comparison of the obtained spectra with a database that can be suitably implemented also modifying the parameters setting, ii) the detection of specific protein biomarkers. For the unique discordant result regarding a E. histolytica strain isolated from a patient with dysentery also positive for E. histolytica antibodies, the presence of amino acid/posttranslational differences as compared to the reference strain could be hypothesized.

MALDI-TOF mass spectrometry as innovative tool applied to parasites identification / Calderaro, Adriana; Montecchini, Sara; Buttrini, Mirko; Rossi, Sabina; Piccolo, Giovanna; Arcangeletti, Maria Cristina; Chezzi, Carlo; DE CONTO, Flora. - (2018), p. 253. ((Intervento presentato al convegno 46° Congresso Nazionale della Società Italiana di Microbiologia tenutosi a Palermo nel 26-29 settembre 2018.

MALDI-TOF mass spectrometry as innovative tool applied to parasites identification

Adriana Calderaro;Sara Montecchini;Mirko Buttrini;Sabina Rossi;Giovanna Piccolo;Maria Cristina Arcangeletti;Carlo Chezzi;Flora De Conto
2018

Abstract

increasingly utilized as a rapid technique to identify microorganisms by their molecular fingerprint and/or by biomarker detection and it represents a first-line method for the accurate routinely identification of bacteria and fungi; its application in parasitology is on the contrary very limited. In this study MALDI-TOF MS was used to identify Trichomonas vaginalis, Dientamoeba fragilis and to differentiate Entamoeba histolytica and E. dispar. Introduction Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is Materials and Methods In this study, an aliquot of one cultured reference strain for each parasite were submitted to formic acid/acetonitril protein extraction and to MALDI-TOF MS analysis. The spectrum obtained for T. vaginalis was suplemented in the Bruker Daltonics database (Bruker Daltonics, Germany) and a new identification method was created by modifying the range setting for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. The spectra obtained for E. histolytica, E. dispar and D. fragilis were analysed and subsequently imported into the ClinProTools software version 2.2. (Bruker Daltonics) to perform a statistical analysis in order to check the presence of specific peaks for each parasite. To verify the reliability of the system 21 T. vaginalis, 6 E. histolytica, 8 E. dispar and 13 D. fragilis clinical isolates, respectively, were used. Results After implementation and modification of the paramenters' setting, the protein spectra of T. vaginalis clinical isolates were correctly identified. Five discriminating peaks between E. histolytica (2 peaks) and E. dispar (3 peaks) and 6 discriminating peaks for D. fragilis were found, respectively. When the spectra belonging to the clinical isolates were analysed, all the identifications matched those obtained by a specific Real-time PCR, except for one E. histolytica strain. Dicussion and Conclusions Although the massive number of entries in the available commercial database, the absence of reference spectra of parasites does not allow their identification. Our study demonstrated that MALDI-TOF MS can be applied to the identification of parasites by using two different approaches: i) the comparison of the obtained spectra with a database that can be suitably implemented also modifying the parameters setting, ii) the detection of specific protein biomarkers. For the unique discordant result regarding a E. histolytica strain isolated from a patient with dysentery also positive for E. histolytica antibodies, the presence of amino acid/posttranslational differences as compared to the reference strain could be hypothesized.
MALDI-TOF mass spectrometry as innovative tool applied to parasites identification / Calderaro, Adriana; Montecchini, Sara; Buttrini, Mirko; Rossi, Sabina; Piccolo, Giovanna; Arcangeletti, Maria Cristina; Chezzi, Carlo; DE CONTO, Flora. - (2018), p. 253. ((Intervento presentato al convegno 46° Congresso Nazionale della Società Italiana di Microbiologia tenutosi a Palermo nel 26-29 settembre 2018.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2851579
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